This invention is directed to compositions comprising soluble divalent and multivalent heterodimeric analogs of proteins that are involved in immune regulation and methods of making and using the same. The high affinity that these complexes have for their cognate ligands enables them to be effective competitors to T cell receptors and MHC molecules normally involved in transplant rejection and autoimmune disease. Molecules such as divalent T cell receptors may also have an impact on the diagnosis and treatment of cancer in that they may be used to augment antitumor responses, or may be conjugated to toxins which may then be used to help eliminate tumors. Use of such compositions will allow one to accomplish selective immune modulation without compromising the general performance of the immune system.
The immune system is a defense system found in most advanced forms of higher vertebrates. A properly functioning lymphatic and immune system distinguishes between self and non-self. A healthy body protects against foreign invaders, such as bacteria, viruses, fungi, and parasites. As the body encounters foreign material (non-self), also known as an antigens, the immune system becomes activated. An antigen is recognized by characteristic shapes or epitopes on its surface. This defense mechanism provides a means of rapid and highly specific responses that are used to protect an organism against invasion by pathogenic microorganisms. It is the myriad of pathogenic microorganisms that have principally caused the evolution of the immune system to its current form. In addition to protection against infectious agents, specific immune responses are thought to be involved in surveillance against alterations in self antigens as seen in tumor development. Immune responses are also involved in the development of autoimmune disease, AIDS, as well as rejection of transplanted tissues.
Lymphocytes
Within the immune system, lymphocytes play a central role. Lymphocyte responses to foreign organisms orchestrate the effector limbs of the immune system, and ultimately, determine the fate of an infection. Lymphocytes can be divided into two main categories, B and T cells. These two types of lymphocytes are specialized in that they have different effector functions and play different roles in the development of specific immune responses. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. Specificity is conferred by an unique set of cell surface receptors expressed on individual lymphocytes. These receptors interact with soluble proteins, in the case of B cells, and with antigenic peptide/major histocompatibility complex (MHC) molecules in the case of T lymphocytes. The nature of the interaction with their ligands also differs between B and T cells. The antigen receptors produced by B cells, immunoglobulins (Igs), interact with their ligands with a high affinity. In contrast, T cell receptors interact with their ligands with low affinity. Thus, the T cell response is driven by the interaction of many T cell receptors (TcR) on the surface of an individual T cell interacting with multiple antigenic peptide/MHC complexes on the surface of the antigen presenting cell. Thus, these two diverse groups of cell-surface glycoproteins, the TCRS and the MHC glycoproteins, form key components of specificity in the T lymphocyte response to antigens.
T cells are a major regulatory cell of the immune system. Their regulatory functions depend not only on expression of a unique T cell receptor, but also on expression of a variety of accessory molecules and effector functions associated with an individual T cell response. Effector functions include responses such as cytotoxic responses or other responses characterized by secretion of effector molecules, i.e., lymphokines. It is this regulatory function that often goes awry in the development of autoimmune diseases. The different effector functions also play a large role in tissue graft rejection, and can be important in tumor rejection.
T cells respond to antigens in the context of either Class I or Class II MHC molecules. Cytotoxic T cells respond mainly against foreign antigens in the context of Class I glycoproteins, such as viral-infected cells, tumor antigens and transplantation antigens. In contrast, helper T cells respond mainly against foreign antigens in the context of Class II molecules. Both types of MHC molecules are structurally distinct, but fold into very similar shapes. Each MHC molecule has a deep groove into which a short peptide, or protein fragment, can bind. Because this peptide is not part of the MHC molecule itself, it varies from one MHC molecule to the next. It is the presence of foreign peptides displayed in the MHC groove that engages clonotypic T cell receptors on individual T cells, causing them to respond to foreign antigens.
Antigen-specific recognition by T cells is based on the ability of clonotypic T cell receptor to discriminate between various antigenic-peptides resident in MHC molecules. These receptors have a dual specificity for both antigen and MHC (Zinkemagel et al., Nature 248, 701-02, 1974). Thus, T cells are both antigen-specific and MHC-restricted. A simple molecular interpretation of MHC-restricted recognition by T cells is that TCRs recognize MHC residues as well as peptide residues in the MHC-peptide complex. Independent of the exact mechanism of recognition, the clonotypic T cell receptor is the molecule that is both necessary and sufficient to discriminate between the multitude of peptides resident in MHC.
T cells can be divided into two broad subsets; those expressing xcex1/xcex2 TcR and a second set that expresses xcex3/xcex4 TcR. Cells expressing xcex1/xcex2 TcR have been extensively studied and are known to comprise most of the antigen-specific T cells that can recognize antigenic peptide/MHC complexes encountered in viral infections, autoimmune responses, allograft rejection and tumor-specific immune responses. Cells expressing xcex1/xcex2 TCRs can be further divided into cells that express CD8 accessory molecules and cells that express CD4 accessory molecules. While there is no intrinsic difference between the clonotypic xcex1/xcex2 T cell receptors expressed either on CD4 and CD8 positive cells, the accessory molecules largely correlate with the ability of T cells to respond to different classes of MHC molecules. Class I MHC molecules are recognized by CD8+, or cytotoxic, T cells and class II MHC molecules by CD4+, or helper, T cells. xcex3/xcex4 T cells make up another significant population of T cells seen in circulation as well as in specific tissues. These cells are not well understood; their antigen/MHC specificity is poorly defined and in most cases their ligands are completely unknown. These cells are present in high quantities in certain tissues, including skin and gut epithelium, and are thought to play a significant role in immune responses of those organs. They have also been implicated in autoimmune responses and may be involved in the recognition of heat shock proteins. A general approach to the identification of antigenic complexes, as outlined in the present invention, would greatly facilitate understanding of how these cells influence the development of both normal and abnormal immune responses. There is a large degree of homology between both xcex1/xcex2 and xcex3/xcex4 TcR expressed in rodents and humans. This extensive homology has, in general, permitted one to develop murine experimental models from which results and implications may be extrapolated to the relevant human counterpart.
MHC Molecules in Health and Disease
Major histocompatibility antigens consist of a family of antigens encoded by a complex of genes called the major histocompatibility complex. In mice, MHC antigens are called H-2 antigens (Histocompatibility-2 antigens). In humans MHC antigens are called HLA antigens (Human-Leukocyte-associated Antigens). The loci that code for MHC glycoproteins are polymorphic. This means that each species has several different alleles at each locus. For example, although a large number of different Class I antigens may be seen in a species as a whole, any individual inherits only a single allele from each parent at each locus, and therefore expresses at most two different forms of each Class I antigen.
In the murine system, the class II MHC molecules are encoded by I-A and I-E loci, and in humans, class II molecules are encoded by the DR, DP and DQ loci. Polymorphism of class II alleles is attributed to the alpha and beta chains and specificities are designated using the nomenclature set forth by the World Health Organization (Immunogenetics 36, 135, 1992).
The Role of MHC Moleculesxe2x80x94Transplantation
MHC molecules play an essential role in determining the fate of grafts. Various species display major immunological functional properties associated with the MHC including, but not limited to, vigorous rejection of tissue grafts, stimulation of antibody production, stimulation of the mixed lymphocyte reaction (MLR), graft-versus-host reactions (GVH), cell-mediated lympholysis (CML), immune response genes, and restriction of immune responses. Transplant rejection occurs when skin, organs (e.g., kidney, liver, lung), or other tissues (e.g., blood, bone marrow) are transplanted across an MHC incompatibility. A vigorous graft rejection occurs when the immune system is activated by mismatched transplantation antigens that are present in donor tissue but not in recipient. Graft rejection may occur in the graft itself by exposure of circulating immune cells to foreign antigens, or it may occur in draining lymph nodes due to the accumulation of trapped transplantation antigens or graft cells. Because of the extensive diversity of MHC antigens, numerous specificities are possible during physiological and pathophysiologic immune-related activities, (e.g., transplantation, viral infections, and tumor development). The recognized HLA specificities are depicted, for example, in a review by Bodmer et al., in Dupont, ed., Immunobiology OF HLA, vol. 1, New York, Springer-Verlag, 1989.
The Role of MHC Moleculesxe2x80x94Autoimmune Response
Susceptibility to many autoimmune disease shows a significant genetic component and familial linkage. Most genetic linkages of autoimmune diseases are with certain class II MHC alleles (see Table 1 for Overview). The level of association between a particular disease and an allele at one of the MHC loci is defined by a term called xe2x80x9crelative risk.xe2x80x9d This term reflects the frequency of the disease in individuals who have the antigen compared to the frequency of the disease among individuals who lack the antigens. For example, there is a strong association with DQxcex2 genotype in insulin-dependent diabetes mellitus; the normal DQxcex2 sequence has an aspartic acid at position 57, whereas in Caucasoid populations, patients with diabetes most often have valine, serine or alanine at that position.
Regulation of Immune Responses
Interest in analyzing both normal and abnormal T cell-mediated immune responses led to the development of a series of novel soluble analogs of T cell receptors and MHC molecules to probe and regulate specific T cell responses. The development of these reagents was complicated by several facts. First, T cell receptors interact with peptide/MHC complexes with relatively low affinities (Matsui et al., Science 254, 1788-891, 1991; Sykulev et al., Immunity 1, 15-22, 1994; Corr et al., Science 265, 946-49 ,1994). To specifically regulate immune responses, soluble molecules with high affinities/avidities for either T cell receptors or peptide/MHC complexes are needed. However, simply making soluble monovalent analogs of either T cell receptors or peptide/MHC complexes has not proven to be effective at regulating immune responses with the required specificity and avidity.
To regulate immune responses selectively, investigators have made soluble versions of proteins involved in immune responses. Soluble divalent analogs of proteins involved in regulating immune responses with single transmembrane domains have been generated by several laboratories. Initially, CD4/Ig chimeras were generated (Capon et al., Nature 337, 525-31, 1989; Bryn et al., Nature 344, 667-70, 1990), as well as CR2/Ig chimeras (Hebell et al., Science 254, 102-05, 1991). Later it was demonstrated that immune responses could be modified using specific CTLA-4/Ig chimeras (Linsley et al., Science 257, 7920-795, 1992; U.S. Pat. No. 5,434,131; Lenschow et al., Science 257, 789-91, 1992). In addition, class I MHC/Ig chimeras were used to modify in vitro allogeneic responses (Dal Porto et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6671-75, 1993). However, these examples include only soluble divalent analogs of single transmembrane polypeptide molecules and not chimeric molecules of heterodimeric proteins in which the heterodimer consists of xcex1 and xcex2 polypeptides that are both transmembrane polypeptides. The present invention reports the generation of soluble divalent and multivalent heterodimeric analogs of integral membrane protein complexes, which consist of alpha and beta polymorphic integral membrane polypeptides that properly fold to form a functional unit that has potential use in immune modulation.
Previously, replacement of two transmembrane domains in the generation of multivalent analogs has not been achieved. The challenge of generating these molecules lies in achieving the proper folding and expression of two polypeptides, both of which ordinarily require transmembrane domains (FIG. 1). In addition, soluble multivalent analogs of heterodimeric proteins generally have increased affinity and, therefore, are preferred therapeutic agents. These soluble protein complexes, which consist of xcex1 and xcex2 polymorphic integral membrane polypeptides that properly fold to form a functional unit, have potential use as immune modulating agents.
Moreover, generation of soluble divalent or multivalent molecular complexes comprising MHC class II or T cell receptors (TCR) is complicated by the fact that such complexes are formed by heterodimeric integral membrane proteins. Each of these protein complexes consists of xcex1 and xcex2 integral membrane polypeptides which bind to each other, forming a functional unit involved in immune recognition. While both class II MHC and TCR molecules have stable, disulfide-containing immunoglobulin domains, obtaining them in properly folded form in the absence of their respective integral membrane regions has proven to be difficult (6, 12).
Strategies have been developed to facilitate subunit pairing and expression of soluble analogs of integral membrane heterodimeric complexes (for review, see 4). Initially, the extracellular domains of a TCR (5, 6) or class II MHC (7) were linked via glycosylphosphatidylinositol (GPI) membrane anchor sequences, resulting in surface expression of the polypeptide chains to enhance subunit pairing. Subsequent enzymatic cleavage resulted in the release of soluble monovalent heterodimers from the GPI anchors. Another strategy facilitated pairing by covalent linkage of immunoglobulin light chain constant regions to constant regions of the TCR xcex1 and xcex2 chains (8). Direct pairing of the xcex1 and xcex2 chains of a TCR during synthesis has also been accomplished by covalent linkage of the variable regions of the xcex1 and xcex2 chains spaced by a 25 amino acid spacer (9) or by linking the variable region of the a chain to the extracellular Vxcex2Cxcex2 chain with a 21 amino acid spacer (10). This strategy, too, results in monomers. In several constructs, xcex1/xcex2 dimerization was facilitated by covalent linkage of the leucine zipper dimerization motif to the extracellular domains of the xcex1 and xcex2 polypeptides of TCR or class II MHC (11-13). Pairing of the extracellular domains of the xcex1 and xcex2 chains of class II MHC has also been achieved after the chains were produced in separate expression systems (14, 15). However, the utility of these probes is limited by their intrinsic low affinity for cognate ligands.
Approaches have also been developed to generate probes for antigen-specific T cells. The first approach used to develop specific reagents to detect clonotypic TCRS was the generation of high affinity anticlonotypic monoclonal antibodies. Anticlonotypic monoclonal antibodies discriminate on the basis of specific TCR Vxcex1 and Vxcex2 conformational determinants, which are not directly related to antigenic specificity. Therefore, an anticlonotypic antibody will interact with only one of potentially many antigen-specific different clonotypic T cells that develop during an immune response.
The development of reagents which differentiate between specific peptide/MHC complexes has also been an area of extensive research. Recently, investigators have used soluble monovalent TCR to stain cells by crosslinking TCRS with avidin after they have been bound to a cell (10). Another approach has been to generate monoclonal antibodies which differentiate between MHC molecules on the basis of peptides resident in the groove of the MHC peptide binding site. While theoretically this approach is appealing, such antibodies have been difficult to generate. Conventional approaches have produced only a few such antibodies with anti-peptide/MHC specificity (36-38). It is not clear why this is the case, but the difficulty may reflect the fact that peptides are generally buried within the MHC molecule.
Two new approaches have been developed to obtain peptide-specific, MHC dependent monoclonal antibodies. One approach utilizes a recombinant antibody phage display library to generate antibodies which have both peptide-specificity and MHC restriction (42). In the second approach, mice are immunized with defined peptide/MHC complexes, followed by screening of very large numbers of the resultant monoclonal antibodies (43, 44). However, the need to screen large numbers of monoclonal antibodies is a disadvantage of this method.
Thus, there is a need in the art for soluble, multivalent molecular complexes with high affinity for antigenic peptides which can be used, for example, to detect and regulate antigen-specific T cells and as therapeutic agents for treating disorders involving immune system regulation.
It is an object of the present invention to provide reagents which specifically and stably bind to and modulate antigen-specific T cells. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides a molecular complex which comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide. The immunoglobulin heavy chain comprises a variable region. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide. The fusion proteins associate to form the molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the first and second transmembrane polypeptides. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein.
Another embodiment of the invention provides a polynucleotide. The polynucleotide encodes a first and a second fusion protein. The first fusion protein comprises an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide of a heterodimeric protein. The immunoglobulin heavy chain comprises a variable region. The immunoglobulin light chain is C-terminal to the extracellular domain of the first transmembrane polypeptide. The second fusion protein comprises an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide of the heterodimeric protein. The immunoglobulin light chain is C-terminal to the extracellular portion of the second transmembrane polypeptide. The extracellular domains of the first and second transmembrane polypeptides form a ligand binding site.
Still another embodiment of the invention provides a host cell comprising at least one expression construct encoding a first and a second fusion protein. The first fusion protein comprises an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide of a heterodimeric protein. The immunoglobulin heavy chain comprises a variable region wherein the immunoglobulin light chain is C-terminal to the extracellular domain of the first transmembrane polypeptide. The second fusion protein comprises an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide of the heterodimeric protein. The immunoglobulin light chain is C-terminal to the extracellular portion of the second transmembrane polypeptide. The extracellular domains of the first and second transmembrane polypeptides form a ligand binding site. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein.
Yet another embodiment of the invention provides a composition. The composition comprises a cell in which a molecular complex is bound to the surface of the cell. The molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular portion of a first transmembrane polypeptide. The immunoglobulin heavy chain comprises a variable region. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular portion of a second transmembrane polypeptide. The fusion proteins associate to form a molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the first and second transmembrane polypeptides. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein.
A further embodiment of the invention provides a method for treating a patient suffering from an allergy. A molecular complex is administered to the patient at a dose sufficient to suppress or reduce a T cell response associated with the allergy. The molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide. The immunoglobulin heavy chain comprises a variable region. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide. The fusion proteins associate to form a molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the first and second transmembrane polypeptides. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein. Each ligand binding site is bound to an antigenic peptide. The antigenic peptide is an antigen to which the patient has an allergic response.
Even another embodiment of the invention provides a method for treating a patient who has received or will receive an organ transplant. A molecular complex is administered to the patient at a dose sufficient to suppress or reduce an immune response to the organ transplant. The molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide. The immunoglobulin heavy chain comprises a variable region. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide. The fusion proteins associate to form a molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the first and second transmembrane polypeptides. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein. Each ligand binding site is bound to an antigenic peptide. The antigenic peptide is an alloantigen.
Yet another embodiment of the invention provides a method for treating a patient suffering from an autoimmune disease. A molecular complex is administered to the patient at a dose sufficient to suppress or reduce the autoimmune response. The molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide. The immunoglobulin heavy chain comprises a variable region. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide. The fusion proteins associate to form a molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the first and second transmembrane polypeptides. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein. Each ligand binding site is bound to an antigenic peptide. The antigenic peptide is one to which the patient expresses an autoimmune response.
Another embodiment of the invention provides a method for treating a patient having a tumor. A molecular complex is administered to the patient at a dose sufficient to induce or enhance an immune response to the tumor. The molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide. The immunoglobulin heavy chain comprises a variable region. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide. The fusion proteins associate to form a molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the first and second transmembrane polypeptides. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein. Each ligand binding site is bound to an antigenic peptide. The antigenic peptide is expressed on the tumor.
Still another embodiment of the invention provides a method for treating a patient having an infection caused by an infectious agent. A molecular complex is administered to the patient at a dose sufficient to induce or enhance an immune response to the infection. The molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide. The immunoglobulin heavy chain comprises a variable region. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide. The fusion proteins associate to form a molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the first and second transmembrane polypeptides. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein. Each ligand binding site is bound to an antigenic peptide. The antigenic peptide is a peptide of the infectious agent.
Another embodiment of the invention provides a method of labeling antigen-specific T cells. A sample which comprises antigen-specific T cells is contacted with a molecular complex. The molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide. The immunoglobulin heavy chain comprises a variable region. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide. The fusion proteins associate to form the molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the first and second transmembrane polypeptides. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein. Each ligand binding site is bound to an identical antigenic peptide. The antigenic peptide specifically binds to the antigen-specific T cells. The cells are labeled with the molecular complex.
Yet another embodiment of the invention provides a method of activating antigen-specific T cells. A sample which comprises antigen-specific T cells is contacted with a molecular complex. The molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular domain of a first transmembrane polypeptide. The immunoglobulin heavy chain comprises a variable region. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide. The fusion proteins associate to form the molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the first and second transmembrane polypeptides. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein. Each ligand binding site is bound to an identical antigenic peptide. The antigenic peptide specifically binds to and activates the antigen-specific T cells.
Even another embodiment of the invention provides a method of labeling a specific peptide/MHC complex. A sample comprising a peptide/MHC complex is contacted with a composition comprising a molecular complex. The molecular complex comprises at least four fusion proteins. Two first fusion proteins comprise an immunoglobulin heavy chain and an extracellular domain of a TCR xcex1 chain. Two second fusion proteins comprise an immunoglobulin light chain and an extracellular domain of a TCR xcex2 chain. The fusion proteins associate to form a molecular complex. The molecular complex comprises two ligand binding sites. Each ligand binding site is formed by the extracellular domains of the TCR xcex1 and xcex2 chains. The affinity of the molecular complex for a cognate ligand is increased at least two-fold over a dimeric molecular complex consisting of a first and a second fusion protein. The ligand binding site specifically binds to and labels the peptide/MHC complex.
Thus, the present invention provides a general approach for producing soluble multivalent versions of heterodimeric proteins, such as T cell receptors and class II MHC molecules. These multivalent molecules can be used, inter alia, as diagnostic and therapeutic agents for treating immune disorders and to study cell-cell interactions which are driven by multivalent ligand-receptor interactions.